Since the advent of humanized antibodies, the therapeutic use of antibodies such as Rituxan® (rituximab), Herceptin® (trastuzumab) and Avastin® (bevacizumab), has revolutionized the fields of medicine, including oncology, the treatment of inflammatory disorders, such as rheumatoid arthritis, and many other indications. In the United States, more than 30 human or humanized antibodies have been approved for clinical use, and more than 600 new antibodies or antibody-like molecules are in various stages of development. Some antibodies have antagonistic function on soluble target molecules such as vascular endothelial growth factor (VEGF) or tumor necrosis factor (TNF), whose actions are part of the pathologic process of a disease. Alternatively, antibodies can bind, block and/or induce destruction of pathologic cells in certain diseases, such as cancer. The main functions of these therapeutic antibodies are binding through the Fab region, and recruitment of effector function via the Fc domain (which also mediates the long circulating half-life of antibodies). One of the major advantages of antibodies compared to small molecule drugs can be their exquisite specificity. Antibodies can very accurately target selected protein antigens, such as oncogenes, to the exclusion of very similar homologs, allowing for benign safety profiles. Hence, antibodies are well characterized for specific single targeting function.
As the field has progressed, antibody function has been enhanced through creative means of protein engineering, such as to provide higher affinity, longer half-life, and/or better tissue distribution, as well as combination of small and large molecule technologies for increased focus of cell destruction via toxic payload delivery (e.g. antibody-drug conjugates). Another approach to improving antibody function takes advantage of the multivalent binding capabilities of the immunoglobulin A (IgA) or immunoglobulin M (IgM) structure which allows one IgA or IgM molecule to bind multiple antigens. Heavy and light chain variable domains of interest can be expressed as an IgA or IgM isotype antibody, thereby creating a multimeric binding molecule with the same specificity as a monomeric antibody, e.g., an IgG antibody.
The multivalent nature of IgA or IgM molecules presents a useful tool for application to specific biological systems in which multiple components necessarily must be bound simultaneously to transmit biological signals. For instance, many receptor proteins on the surface of eukaryotic cells require the simultaneous activation of multiple monomers or subunits to achieve activation and transmission of a biological signal across a cell membrane, to the cytoplasm of the cell.
One such system of cell surface protein receptors requiring multimerization prior to, or commensurate with, activation is found in the Tumor Necrosis Factor (TNF) superfamily of receptor proteins. Within this superfamily of receptor proteins are members which, upon activation, transmit a signal to the nucleus of the cell causing apoptosis. Other family members of this superfamily cause activation of NF-κB, apoptosis pathways, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), and c-Jun N-terminal kinase (JNK). Examples of TNF superfamily receptor members which regulate apoptosis of the cell when activated are the following: TNFR1 (DR1), TNFR2, TNFR1/2, CD40 (p50), Fas (CD95, Apo1, DR2), CD30, 4-1BB (CD137, ILA), TRAILR1 (DR4, Apo2), DR5 (TRAILR2), TRAILR3 (DcR1), TRAILR4 (DcR2), OPG (OCIF), TWEAKR (FN14), LIGHTR (HVEM), DcR3, DR3, EDAR, and XEDAR. (See, Aggarwal et al., Blood, 119:651-665, 2012).
More particularly, it is postulated that activation of the TNF superfamily receptor protein members mentioned above requires that at least three non-interacting receptor monomers be cross-linked, e.g., by a ligand, to form a stabilized receptor trimer, resulting in signal transduction across the cell membrane. Clustering of these TNF superfamily receptor protein trimers into “rafts” of trimers has been observed and has been postulated to lead to more effective activation of this TNF superfamily receptor protein-dependent signaling cascade. (See, Valley et al., J. Biol. Chem., 287(25):21265-21278, 2012). Additional modes of activation have been discussed. (See, for instance, Lewis et al., Biophys. J., 106(6):L21-L24, 2014).
Signaling through certain of the TNF superfamily receptor proteins noted above can lead to cell apoptosis. In the treatment of cancer, one therapeutic strategy is to activate an apoptotic signaling cascade in cancer cells, thereby halting progression. One manner in which this can be accomplished is by the binding of TNF superfamily receptor proteins expressed (or over-expressed) in cancer cells with a multivalent or multimeric agonist binding molecule, which can promote receptor trimerization and activation, leading to apoptosis. One TNF superfamily receptor protein that is activated upon cross-linking resulting in apoptosis is DR5 (TRAILR2).
Interest—in DR5 is heightened due to the finding that it is expressed at a higher level in various cancers than in normal tissue, such as bladder cancer (Y et al., Urology, 79(4):968.e7-15, 2012), gastric cancer (Lim et al., Carcinogen., 32(5):723-732, 2011), ovarian cancer (Jiang et al., Mol. Med. Rep., 6(2):316-320, 2012), pancreatic ductal adenocarcinoma (Rajeshkumar et al., Mol. Cancer Ther., 9(9):2583-92, 2010), oral squamous cell carcinoma (Chen et al. Oncotarget 4:206-217, 2013) and non-small cell lung cancer (Reck et al., Lung Canc., 82(3):441-448, 2013). It is of additional importance to the medical community that the observed higher level of expression of this family of receptor proteins, especially family member DR5, occurs in some of the most difficult to detect and treat cancers, such as pancreatic and gastric cancer.
While certain monoclonal antibodies, such as Tigatuzumab (CS-1008, Daiichi Sankyo Co. Ltd., disclosed in U.S. Pat. No. 7,244,429, VH and VL presented herein as SEQ ID NO: 7 and SEQ ID NO: 8, respectively), have been found to be effective in vitro and in vivo even without additional cross-linkers added, these antibodies have not resulted in significant clinical efficacy. (See, Reck et al., 2013). Examples of such anti-DR5 agonistic monoclonal IgG antibodies are Conatumumab (Amgen, described in U.S. Pat. No. 7,521,048, VH and VL presented herein as SEQ ID NO: 5 and SEQ ID NO: 6, respectively), Drozitumab (Genentech, as described in U.S. Pat. No. 8,029,783, VH and VL presented herein as SEQ ID NO: 3 and SEQ ID NO: 4, respectively), and Lexatumumab (Human Genome Sciences, as disclosed in U.S. Patent Application Publication No. 2006/0269555, VH and VL presented herein as SEQ ID NO: 1 and SEQ ID NO: 2, respectively).
Better binding molecules are needed to achieve the benefits of the basic research performed which provided a critical understanding of this subset of the TNF superfamily receptor proteins. Additional binding molecules are disclosed herein which, based on the understanding of the underlying biochemical mechanism of the TNF superfamily of receptor proteins, are capable of addressing this need.